Screening Isolates Capable Of De-epoxidating Trichothecenes And Exploring The Potential Mechanisms

Trichothecenes are secondary metabolites produced by fungi such as Fusarium graminearum,Myrothecium,Stachybotrys.Trichothecenes are stable in nature and widely contaminate food crops or feed ingredients such as wheat and corn,which not only cause serious economic losses,but also threaten the health of animals and humans seriously.Because of bad effects and possibility secondary pollution in physical and chemical detoxification methods,biological methods have been paid more attention with the characters of high efficiency and environmentally friendly.The de-epoxidation metabolism of trichothecenes is considered to be an effective way to detoxify which most happens in animal intestine or ruminant rumen.However,the isolates capable of de-epoxidating trichothecenes are limited and the potential mechanism has not been reported.Therefore,it is urgent to screen functional microbes from animal intestine or rumen and discover the relative genes involved in de-epoxidating which may solve the current situations of trichothecene-toxin-contaminated food.It also provides a theoretical basis to apply the functional microbes or relative enzymes into feed detoxification.Raoultibacter sp.DⅡ-9(DⅡ-9)which could de-epoxidize at least four trichothecenes,was selected from chicken intestinal at the earlier time.It was necessary to continue screening more isolates capable of de-epoxidating and provide more candidate strains for detoxification in the food industry.And it was also important to illuminate the mechanism of de-epoxidation.During the process,deoxynivalenol(DON)was chose to be as the substrate to detect and conform because of its’widely contaminated in crops or feed.Twenty samples from chicken intestines were used to screen isolates capable of de-epoxidizing DON.Luckily,one isolate was got from more than 1000 clones.It was a member of genus Slackia and was named as Slackia sp.D-G6(D-G6).D-G6 belonged to Gram-positive anaerobic bacteria and had a cell size of about 0.2-0.4μm×0.6-1.0μm.The optimum conditions for the degradation of DON were 37-47°C,p H 6-10 neutral alkaline environment.At optimal conditions,5μg of DON can be completely converted during 1day.Comparing the efficiency of D-G6 and DⅡ-9 in degrading DON,it was found that both of them can degrade 40-50μg of DON in 2 days,and the ability was equivalent.In addition to its function of degrading DON,D-G6 could also convert soybean isoflavone(Daidzein)to equol like other strains of Slackia.Equol can effectively inhibit a variety of estrogen or age-related diseases.Based on the above two biological functions,D-G6 had two potentials.On the one hand,it could be developed into probiotics to improve the intestinal micro-environment which would be beneficial to human health.On the other hand,it can be prepared as a detoxifying agent for the feed industry.The discovery of D-G6 was helpful to clarify the mechanism of trichothecenes de-epoxidation.In order to screen the target gene of degrading trichothecenes,firstly,by comparing genomics,four genomic sequences were analyzed.Two of them,DG-6 and DⅡ-9,have DON de-epoxidation activity.Two of them,DZE~Tand P3277~Twere the closest strain of DG-6 and DⅡ-9,respectively,but with no DON de-epoxidation activity.Three group genes,No.892 and No.2459 and No.4328,were specifically included in the genomes of DⅡ-9 and D-G6 but not in P3277~Tand DZE~T.Secondly,a cDNA library of DⅡ-9 was constructed in Saccharomyces cerevisiae sensitive to DON.The transformed cells of Saccharomyces Cerevisiae were screened at the pressure of trichothecenes.The candidate genes could be got by sequencing.One mutant sensitivity to DON was constructed,and the phenotype was BY4742(MATα)pdr5Δ::Hygromycin pdr10Δ::his3 ayt1Δ::Kan MX ubi4Δ::his3.The growth of the mutant with four genes knockout was completely inhibited when DON was more than 100μg/m L at 30°C.So,the mutant can be taken as a sensitive bioassay indicator organism for DON.At last,four positive transformants tolerant to DON were got,#14、#21、#23、#24.However,it was found that DON resistance resume was not related with the transferred plasmids.Thirdly,a mutant library of DⅡ-9 was constructed using the chemical mutagen ethyl sulfonate(EMS).Five mutants were obtained from more than 30,000 mutants,which greatly reduced the efficiency of degradation of DON compared to WT.Four candidate genes#127,#716,#748,#1695 were located by second-generation sequencing.Their functions are mostly related to material transport and redox reactions.In this study,a microorganism was successfully screened which could degrade DON belonging to the genus Slackia,and named Slackia sp.D-G6.A defective yeast strain sensitive to DON was obtained from the constructed cDNA library,which could be used as a bio-detector for candidate gene verification.Its genome was sequenced.About de-epoxidating trichothecene,10 candidate genes were obtained by three methods.Six of them were labeled as redox functional proteins,and 4 functions were unknown.Further exploration of these 10 candidate genes is needed in vitro.